Histatins and method of use thereof

ABSTRACT

Synthetic histatins composed of combinations of functional domains of natural histatins separated by exogenous linkers are described as are methods of using endogenous and synthetic histatins for the treatment of ocular diseases or conditions.

INTRODUCTION

This application is a continuation of U.S. Ser. No. 15/775,901 filed May14, 2018, which is the National Stage of International Application No.PCT/US2016/063906 filed Nov. 29, 2016, which claims benefit of priorityto U.S. Provisional Patent Application Ser. No. 62/260,705, filed Nov.30, 2015, the contents of which are incorporated herein by reference intheir entirety.

This invention was made with government support under grant number5K08EY024339 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND

Histatins (HTNs) are small histidine-rich cationic peptides found insaliva, as well as human lacrimal epithelium (Aakalu, et al. (2014)Invest. Ophthalmol. Vis. Sci. 55:3115; Ubels, et al. (2012) Invest.Ophthalmol. Vis. Sci. 53(11):6738-47; Steele, et al. (2002) Invest.Ophthalmol. Vis. Sci. 43:98). Histatins range in size from 7 to 38 aminoacid residues in length and represent a group of antimicrobial peptideswith antibacterial properties and significant antifungal properties. Inaddition, histatins have been implicated in wound healing, metal ionchelation, anti-inflammatory effects and angiogenesis (Melino, et al.(2014) FEBS J. 281:657-72; Oudhoff, et al. (2008) FASEB J.22(12):3805-12); Oudhoff, et al. (2009) J. Dent. Res. 88(9):846-50; WO2007/142381). Structure-function studies have identified distinctN-terminal and C-terminal domains in both HTN1 and HTN3, whichrespectively contribute to the antimicrobial and wound healingproperties (Melino, et al. (1999) Biochemistry 38:9626-33; Brewer, etal. (1998) Biochem. Cell Biol. 76:247-56; Gusman, et al. (2001) Biochim.Biophys. Acta 1545:86-95). In this respect, histatins, as well asfragments, multimers and combinations thereof, have been suggested foruse in treating various conditions including ocular surface disease (US2013/0310327; 2013/0310326; WO 2016/060916; WO 2016/060917; WO2016/060918; WO 2016/060921; US 2016/0279194) and wounds (US2013/0288964; US 2011/0178010).

Cyclic analogs of histatins have also been described. For example, U.S.Pat. No. 6,555,650 describes cyclic analogues of HTN5 with disulfidebridges that create a cyclic portion of from 5-16 of said amino acidunits. In addition, head-to-tail cyclization of HTN5 has been shown toincrease amphipathicity of the peptide without affecting itsantimicrobial potency (Sikorska & Kamysz (2014) J. Pept. Sci. 20:952-7).Further, cyclization of histatin-1 has been shown to potentiate themolar activity approximately 1000-fold (Oudhoff, et al. (2009) FASEB J.23:3928-35) and increases wound closure activity (Bolscher, et al.(2011) FASEB J. 25:2650-8). Moreover, cyclic analogs of histatin, withenhanced potency have been suggested for use in treating microbialinfection (US 2010/0173833; Brewer & Lajoie (2002) Biochemistry41:5526-5536).

SUMMARY OF THE INVENTION

This invention provides a synthetic histatin having the generalstructure:[HTNF₁-L₁-HTNF₂-(L₂)_(y)]_(x)  (Formula I)wherein

i) HTNF₁ is a first histatin fragment ranging in length from 5 to 20amino acids;

ii) HTNF₂ is a second histatin fragment ranging in length from 5 to 20amino acids;

iii) L₁ is a first linker;

iv) L₂ is a second linker;

v) x=1 to 3; and

vi) y=0 to 2;

wherein HTNF₁ and HTNF₂ are each independently the same or different andL₁ and L₂ are each independently the same or different. In oneembodiment, HTNF₁ or HTNF₂ has an amino acid sequence of SNYLYDN (SEQ IDNO:1) or HEXXH (SEQ ID NO:2), wherein each X is independently a basicamino acid residue. In another embodiment, L₁ or L₂ is a flexiblelinker, rigid linker, in vivo cleavable linker, or a combinationthereof. In some embodiments, the flexible linker is a hydrocarbonlinker having, e.g., the structure —(CH₂)₆—, or a peptide linker havingan amino acid sequence of (GGGGS)_(n) (SEQ ID NO:3), KESGSVSSEQLAQFRSLD(SEQ ID NO:4), or EGKSSGSGSESKST (SEQ ID NO:5), GGGGGGGG (SEQ ID NO:6),GSAGSAAGSGEF (SEQ ID NO:7), (GGSG)_(n) (SEQ ID NO:8), or (GS)_(n) (SEQID NO:9), wherein n is 1-5. In other embodiments, the rigid linker is apeptide linker having an amino acid sequence of (EAAAK)_(n) (SEQ IDNO:10), A(EAAAK)_(n)A (SEQ ID NO:11), PAPAP (SEQ ID NO:12), or (XP)_(n)(SEQ ID NO:13), wherein n is 1, 2, 3, 4 or 5. In yet another embodiment,the in vivo cleavable linker is a peptide linker having an amino acidsequence of VSQTSKLTRAETVFPDV (SEQ ID NO:14), PLGLWA (SEQ ID NO:15),RVLAEA (SEQ ID NO:16), EDVVCCSMSY (SEQ ID NO:17), GGIEGRGS (SEQ IDNO:18), TRHRQPRGWE (SEQ ID NO:19), AGNRVRRSVG (SEQ ID NO:20), RRRRRRRRR(SEQ ID NO:21), GFLG (SEQ ID NO:22), or CRRRRRREAEAC (SEQ ID NO:23). Instill further embodiments, the synthetic histatin is linear or cyclized,e.g., via a disulfide bridge between terminal cysteine residues or witha sortase or butelase. In certain embodiments, the synthetic histatinhas the structure:

GYKRKFHEKHHSHR (SEQ ID NO:24)-L₁-YGDYGSNYLYDN (SEQ ID NO:25);

HEKHH (SEQ ID NO:26)-L₁-HEKHH (SEQ ID NO:26)-L₂-HEKHH (SEQ IDNO:26)-L₁-YGDYGSNYLYDN (SEQ ID NO:25);

HEKRHH (SEQ ID NO:27)-L₁-HEKRHH (SEQ ID NO:27)-L₂-HEKRHH (SEQ IDNO:27)-L₁-YGDYGSNYLYDN (SEQ ID NO:25);

HEKRHH (SEQ ID NO:27)-L₁-HEKRHH (SEQ ID NO:27)-L₂-HEKHH (SEQ IDNO:26)-L₁-YGDYGSNYLYDN (SEQ ID NO:25); or

HEKRHH (SEQ ID NO:27)-L₁-HEKHH (SEQ ID NO:26)-L₂-HEKHH (SEQ IDNO:26)-L₁-YGDYGSNYLYDN (SEQ ID NO:25), wherein each L₁ and L₂ isindependently a flexible linker, rigid linker, or in vivo cleavablelinker. In specific embodiments, the synthetic histatin has thestructure: GYKRKFHEKHHSHR-(CH₂)₆-YGDYGSNYLYDN (SEQ ID NO:28),HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-YGDYGSNYLYDN (SEQ ID NO:29),HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-YGDYGSNYLYDN (SEQ ID NO:30),HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKHH-(CH₂)₆-YGDYGSNYLYDN (SEQ ID NO:31) orHEKRHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-YGDYGSNYLYDN (SEQ ID NO:32).

A composition containing one or more synthetic histatins of Formula Iand a pharmaceutically acceptable carrier or excipient is also providedas is a kit and methods for treating an ocular disease or condition orproviding wound healing, antimicrobial, metal ion chelating,anti-inflammatory, anti-angiogenic, or matrix metalloproteinaseinhibitory activity in a tissue or organ using one or more synthetichistatins of Formula I.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 show zymographic analysis of synthetic histatin inhibitoryactivity. Experiments included pro-MMP2 (100 ng; “−Ve”); activation ofPro-MMP2 with APMA (1 mM; “+Ve”); inclusion of a classic matrixmetalloproteinase inhibitor, GM6001 (200 nM; “Inh”); and varying amountsof a synthetic histatin containing three metal binding domains fromendogenous HTN5 and a wound healing domain from endogenous HTN1 (“H-1”).The protein ladder is shown on the right.

FIG. 2 shows VEGF-dependent inhibition of angiogenesis by endogenousHTN1, endogenous HTN2 and a synthetic histatin.

FIG. 3 shows the released amount of lactate dehydrogenase (LDH) fromhuman corneal epithelial cells treated with 0.002% benzalkonium chloride(BAK), or native histatin (HTN5) or synthetic HTN (SYN HTN) in thepresence or absence of BAK. Columns represent the mortality (mean±SD) ofcells, total cellular death being 100% (Control).

DETAILED DESCRIPTION OF THE INVENTION

Histatins are appealing molecules for the development of oculartherapeutics because they are amphipathic, which is necessary fortopical application and penetration into the eye, and promote woundhealing through EGF-independent mechanisms. In addition, histatinspromote wound healing without proliferation, inhibit MMP2/MMP9 andangiogenesis, prevent infection, and are anti-inflammatory. Cornealwounds, in particular, can cause vision loss primarily due toinflammatory scarring, and angiogenic vessel invasion. Utilizinghistatin as a wound healing and anti-angiogenic agent has broadimplication for ocular surface disease therapies. It has now been foundthat combinations of functional domains of histatin separated byexogenous linkers provide fusion peptides tailored for use in thetreatment of human ocular disease. In particular, peptides composed ofone or more zinc binding domains, with or without one or more woundhealing domains, and with or without cyclization can be designed tomanipulate the function and efficacy of histatin in various assays andapplications.

Accordingly, this invention provides synthetic histatins and use of suchpeptides and/or endogenous histatins in antimicrobial, wound healing,metal ion chelating, anti-inflammatory, anti-cancer, anti-angiogenic,and/or matrix metalloproteinase inhibitory applications in particular inthe treatment of an ocular disease or condition. In addition, novel usesof endogenous histatin peptides are provided including use in ocularsurface disease, ocular neovascularization and dry eye disease. Asynthetic histatin of this invention has the general structure:[HTNF₁-L₁-HTNF₂-(L₂)_(y)]_(x)  (Formula I)wherein

i) HTNF₁ is a first histatin fragment ranging in length from about 5 toabout 20 amino acids;

ii) HTNF₂ is a second histatin fragment ranging in length from about 5to about 20 amino acids;

iii) L₁ is a first linker;

iv) L₂ is a second linker;

v) x=1 to 3; and

vi) y=0 to 2;

wherein HTNF₁ and HTNF₂ are each independently the same or different andL₁ and L₂ are each independently the same or different.

The term “about” encompasses amounts that differ by ±1-2. Whether or notmodified by the term “about”, the claims include equivalents to thequantities. Where present, all ranges are inclusive and combinable. Forexample, when a range of “1 to 5” is recited, the recited range shouldbe construed as including ranges “1 to 4,” “1 to 3,” “1-2,” “1-2 & 4-5,”“1-3 & 5,” and the like.

A “histatin fragment” or “HTNF” refers to a fragment of a naturalhistatin. A “histatin fragment” or “HTNF” ranges in length from about 5to about 20 amino acid residues and includes a metal binding domain,wound healing domain, or antimicrobial domain of a natural histatin. Incertain embodiments, a histatin fragment is 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues in length. In otherembodiments, a histatin fragment ranges in length from to 5 to 20 aminoacid residues, 6 to 14 amino acid residues, or 12 to 14 amino acidresidues.

In some embodiments, a synthetic histatin is composed of 2 HTNFs, i.e.,x=1 and y=0. In other embodiments, a synthetic histatin is composed of 4HTNFs, i.e., x=2 and y=1. In further embodiments, a synthetic histatinis composed of 6 HTNFs, i.e., x=3 and y=2. Larger synthetic histatinmolecules including, e.g., 8, 10, 12, 14, 16 or more HTNFs, and thoseincluding, e.g., 3, 5, 7, 9, 11, 13, 15 or more HTNFs are alsoconsidered within the scope of this invention. However, it is desirablethat the length the synthetic peptide is in the range of 20 to 50 aminoacid residues.

As used herein, “endogenous histatin,” “native histatin” or “naturalhistatin” refers to a 7-44 amino acid residue, histidine-rich peptide,which was originally identified in saliva and characterized based uponits fungistatic effects. See, e.g., Melino, et al. (2014) FEBS J.281:657-682, and references cited therein. Representative naturalhistatins include, but are not limited to, the peptides listed in Table1.

TABLE 1 SEQ ID Histatin Sequence NO: HTN1 DSpHEKRHHGYRRKFHEKHHSHREFPF 33YGDYGSNYLYDN HTN2 RKFHEKHHSHREFPFYGDYGSNYLYDN 34 HTN3DSHAKRHHGYKRKFHEKHHSHRGYRSN 35 YLYDN HTN4 KFHEKHHSHRGYRSNYLYDN 36 HTN5DSHAKRHHGYKRKFHEKHHSHRGY 37 HTN6 DSHAKRHHGYKRKFHEKHHSHRGYR 38 HTN7RKFHEKHHSHRGY 39 HTN8 KFHEKHHSHRGY 40 HTN9 RKFHEKHHSHRGYR 41 HTN10KFHEKHHSHRGYR 42 HTN11 KRHHGYKR 43 HTN12 KRHHGYK 33 HTN YGDYGSNYLYDN 25C-ter “Sp” or “S(PO₃)” denotes phosphorylated serine.

The term “metal binding domain,” as used herein, refers to an amino acidmotif of natural histatin that binds or forms a complex with a metal.Structural and functional characterization of HTNs revealed the presenceof two metal-binding motifs: the amino-terminal Cu(II)/Ni(II) binding(ATCUN) motif with one histidine residue in the third position(NH₂—X₁X₂H, wherein X₁ is Asp or Glu, and X₂ is Ala, Thr, Met or Ser)(Grogan, et al. (2001) FEBS Lett. 491:76-80; Melino, et al. (2006)Biochemistry 45:15373-83; Melino, et al. (1999) Biochemistry 38:9626-33;Gusman, et al. (2001) Biochim. Biophys. Acta 1545:86-95); and theZn(II)-binding motif HEXXH (SEQ ID NO:2), wherein X denotes a basicamino acid residue such as Lys, Arg, or His. Accordingly, in someembodiments, the synthetic histatin includes an amino-terminal metalbinding domain having the sequence DSH, ESH, DAH, EAH, DTH, ETH, DMH orEMH. In another embodiment, the synthetic histatin includes one or moremetal binding domains having the sequence HEKKH (SEQ ID NO:45), HEKRH(SEQ ID NO:46), HEKHH (SEQ ID NO:26), HERKH (SEQ ID NO:47), HERRH (SEQID NO:48), HERHH (SEQ ID NO:49), HEHKH (SEQ ID NO:50), HEHRH (SEQ IDNO:51) or HEHHH (SEQ ID NO:52). HTNF₁ or HTNF₂ can include the specificsequence of the above-referenced metal binding domains or can includebetween 1 and 6 additional native histatin amino acid residues on the C-and/or N-terminus of the metal binding domain. By way of illustration, aHTNF with a metal binding domain can have the sequence GYKRKFHEKHHSHR(SEQ ID NO:24) or HEKRHH (SEQ ID NO:27)

In some embodiments, a synthetic histatin includes one metal bindingdomain, i.e., either HTNF₁ or HTNF₂ is a metal binding domain. In otherembodiments, a synthetic histatin includes two metal binding domains. Infurther embodiments, a synthetic histatin includes three metal bindingdomains. In certain embodiments, a metal binding domain has the sequenceDHX, wherein X is Ser. In other embodiments, a metal binding domain hasthe sequence HEXXH (SEQ ID NO:2), wherein each X is a basic amino acidresidue. As would be readily appreciated by those of skill in the art,the inclusion of one or more metal binding domains in a synthetichistatin impart metal ion chelating, anti-inflammatory, matrixmetalloproteinase inhibitory, and/or anti-angiogenic activity to thesynthetic histatin. In light of its anti-angiogenic activity, such asynthetic and/or endogenous histatin would be of use in treatingage-related macular degeneration, diabetic retinopathy, cancer, andchronic or acute sever uveitis. In light of its metal ion chelatingactivity, such a synthetic and/or endogenous histatin would also be ofuse in inhibiting tissue destruction mediated by matrixmetalloproteinases and other metal-dependent enzymes in inflammatory andinfectious diseases such as infectious keratitis, intraocular uveitis,endophthalmitis, inflammatory keratitis, dry eye disease and ocularsurface or intraocular diseases.

As used herein, “wound healing domain” refers to an amino acid motif ofnatural histatin that promotes or facilitates wound healing. It has beenreported that HTN1 and HTN3, but not HTN5, exhibit wound closureactivities in vitro (Oudhoff, et al. (2008) FASEB J. 22:3805-3812). Theinactivity of HTN5, which is composed of the N-terminal 22 amino acidresidues of HTN1 and HTN3, indicates that the C-terminal residues inHTN1 and HTN3 are responsible for wound closure activity. Accordingly,for the purposes of this invention, a wound healing domain of naturalhistatin includes the sequence SNYLYDN (SEQ ID NO:1). HTNF₁ or HTNF₂ caninclude the specific sequence of the above-referenced wound healingdomain or can include between 1 and 6 additional native histatin aminoacid residues on the C- and/or N-terminus of the wound healing domain.By way of illustration, a HTNF with a wound healing domain can have thesequence GYKRKFHEKHHSHR (SEQ ID NO:24).

In some embodiments, a synthetic histatin includes one wound healingdomain, i.e., HTNF₁ or HTNF₂ is a wound healing domain. In otherembodiments, a synthetic histatin includes two wound healing domains. Infurther embodiments, a synthetic histatin includes three wound healingdomains. As would be readily appreciated by those of skill in the art,the inclusion of one or more wound healing domains in a synthetichistatin impart epithelial cell migration and spreading activity to thesynthetic histatin. Such a synthetic histatin would therefore be of usein wound healing as well as the treatment of retinal pigment epithelialhealing, dry age-related macular degeneration, ocular surface diseasesand ocular surface inflammatory disorders, ocular neovascularizationincluding corneal and intraocular, retinal or choroidal, and dry eyediseases.

The term “antimicrobial domain,” as used herein, refers to an amino acidmotif of natural histatin that exhibits cytostatic or cytocidal activitytoward bacterial and/or fungal cells. Characterization of HTNs indicatesthat a positive net charge and the amino-terminal portion of HTNsmediate antimicrobial activity. In particular, the amino acid sequenceRKFHEKHHSHRGYR (SEQ ID NO:53) of HTN3 has been shown to exhibitfungicidal activity (Oppenheim, et al. (2012) PLoS ONE 7(12):e51479).Similarly, the sequence AKRHHGYKRKFH (SEQ ID NO:54), also known asP-113, exhibits fungicidal activity against Candida albicans (Jang, etal. (2008) Antimicrob. Agents Chemother. 5292):497-504). HTNF₁ or HTNF₂can include the specific sequence of the above-referenced antimicrobialdomains or can include between 1 and 6 additional native histatin aminoacid residues on the C- and/or N-terminus of the wound healing domain.

In some embodiments, a synthetic histatin includes one antimicrobialdomain, i.e., HTNF₁ or HTNF₂ is an antimicrobial domain. In otherembodiments, a synthetic histatin includes two antimicrobial domains. Infurther embodiments, a synthetic histatin includes three antimicrobialdomains. In certain embodiments, an antimicrobial domain has thesequence RKFHEKHHSHRGYR (SEQ ID NO:53). In other embodiments, anantimicrobial domain has the sequence AKRHHGYKRKFH (SEQ ID NO:54). Aswould be readily appreciated by those of skill in the art, the inclusionof one or more antimicrobial domains in a synthetic histatin impartantifungal and/or antibacterial activity to the synthetic histatin. Sucha synthetic histatin would therefore be of use in treating microbialinfections such as Candida eye infection as well as preventinginfections associated with surgical implants.

Each of the individual histatin fragments, i.e., HTNF₁ and HTNF₂, of thesynthetic histatin of this invention can be the same or different. Insome embodiments, a synthetic histatin is composed of at least one metalbinding domain and at least one wound healing domain. Accordingly, inembodiments of Formula I where x is 1 and y is 0, HTNF₁ is a metalbinding domain and HTNF₂ is a wound healing domain. In embodiments ofFormula I where x is 2 and y is 1, each HTNF₁ is a metal binding domainand each HTNF₂ is a wound healing domain. In other embodiments where xis 2 and y is 1, each HTNF₁ is a metal binding domain, one HTNF₂ is ametal binding domain and one HTNF₂ is a wound healing domain. In furtherembodiments, HTNF₁ and HTNF₂ are individually derived from the same ordifferent natural histatins (i.e., HTN1-HTN12). For example, a synthetichistatin can be composed of one or more metal binding domains from HTN5and a wound healing domain from HTN1. Alternatively, a synthetichistatin can be composed of one or more metal binding domains from HTN1and a wound healing domain from HTN1. Examples of synthetic histatinscontaining combinations of metal binding domains and wound healingdomains are presented in Table 2.

TABLE 2 Synthetic Histatin GYKRKFHEKHHSHR(SEQ ID NO: 24)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25)HEKHH(SEQ ID NO: 26)-L₁-HEKHH(SEQ ID NO: 26)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25)HEKRHH(SEQ ID NO: 27)-L₁-HEKRHH(SEQ ID NO: 27)-L₂-HEKRHH(SEQ ID NO: 27)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25)HEKRHH(SEQ ID NO: 27)-L₁-HEKRHH(SEQ ID NO: 27)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25)HEKRHH(SEQ ID NO: 27)-L₁-HEKHH(SEQ ID NO: 26)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25)

As used herein, the terms “linker” or “spacer” refers to a heterologousmolecule used to connect, link or join together two or more HTNFs. Theterm “heterologous molecule” refers to a molecule that is not normallyfound in a histatin or not typically disposed between the HTNF aminoacid sequences in nature. As used herein, the term “linked,” “joined” or“connected” generally refers to a functional linkage between twocontiguous or adjacent amino acid sequences to produce a molecule thatdoes not exist in nature. Generally, the linked amino acid sequences arecontiguous or adjacent to one another and retain their respectiveoperability and function when joined. The linkers may provide desirableflexibility to permit the desired expression, activity and/orconformational positioning of the synthetic histatin.

In some embodiments, a synthetic histatin is composed of 2 HTNFs and onelinker, i.e., x=1 and y=0. In other embodiments, a synthetic histatin iscomposed of 4 HTNFs and three linkers, i.e., x=2 and y=1. In furtherembodiments, a synthetic histatin is composed of 6 HTNFs and fivelinkers, i.e., x=3 and y=2. However, it is contemplated that additionallinkers can be used when more HTNFs are combined.

Linkers of use in the synthetic histatin of Formula I can be flexible,rigid, in vivo cleavable, or a combination thereof. In addition, linkerscan be composed of amino acid residues (i.e., peptide linkers) orcomposed of chains of hydrocarbons (i.e., hydrocarbon linkers). Peptidelinkers can be of any appropriate length to connect one or more HTNFs ofinterest and are preferably designed so as to allow the proper foldingand/or function and/or activity of one or both of the HTNFs it connects.Thus, the linker peptide can have a length of no more than 3, no morethan 5, no more than 10, no more than 15, no more than 20, no more than25, no more than 30, no more than 35, no more than 40, no more than 45,no more than 50, no more than 55, or no more than 60 amino acids. Insome embodiments, the linker peptide can have a length of at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 12, at least 15, at least 18, at least 20, at least25, at least 30, at least 35, at least 40, at least 45, or at least 50amino acids. In some embodiments, the linker includes at least 10 and nomore than 60 amino acids, at least 10 and no more than 55 amino acids,at least 10 and no more than 50 amino acids, at least 10 and no morethan 45 amino acids, at least 10 and no more than 40 amino acids, atleast 10 and no more 35 amino acids, at least 10 and no more than 30amino acids, at least 10 and no more than 25 amino acids, at least 10and no more than 20 amino acids or at least 10 and no more than 15 aminoacids.

A “flexible” linker refers to a hydrocarbon or peptide linker that doesnot have a fixed structure (secondary or tertiary structure) insolution. Such a flexible linker is therefore free to adopt a variety ofconformations. Flexible linkers of use herein include hydrocarbonlinkers and peptide linkers composed of small, non-polar (e.g., Gly)and/or polar (e.g., Ser or Thr) amino acid residues. Simple amino acids(e.g., amino acids with simple side chains (e.g., H, CH₃ or CH₂OH) areadvantageous for use in a peptide linker as the lack of branched sidechains on these amino acids provides greater flexibility (e.g.,two-dimensional or three-dimensional flexibility) within the linker and,accordingly, within a polypeptide composition. The flexible linker maycontain additional amino acids such as Thr and Ala to maintainflexibility, as well as polar amino acids such as Lys and Glu to improvesolubility. The amino acids can alternate/repeat in any mannerconsistent with the linker remaining functional (e.g., resulting inexpressed and/or active polypeptide(s)). Flexible linkers are described,for example, in Chen, et al. (2013) Adv. Drug Deliv. Rev.65(10):1357-1369; US 2012/0232021; US 2014/0079701; WO 1999/045132; WO1994/012520 and WO 2001/1053480.

In other embodiments, the flexible linker is a hydrocarbon linker. Thehydrocarbon linking the HTNFs should have sufficient length andflexibility so that the synthetic histatin can achieve the desiredconformation. In certain embodiments, the hydrocarbon is composed of oneor more methylene (—CH₂—) groups. In certain embodiments, thehydrocarbon includes between 3 and 25 methylene groups, i.e.,—(CH₂)_(n)—, wherein n is 3 to 25. In certain embodiments, thehydrocarbon linker has the structure —(CH₂)₆—. Additional carbon-basedlinkers such as glycol linkers could also be used in the synthetichistatin of this invention.

In other embodiments, the linker is a rigid linker. “Rigid” linkerrefers to a molecule that adopts a relatively well-defined conformationwhen in solution. Rigid linkers are therefore those which have aparticular secondary and/or tertiary structure in solution. Rigidlinkers are typically of a size sufficient to confer secondary ortertiary structure to the linker. Such linkers include aromaticmolecules (see, e.g., U.S. Pat. Nos. 6,096,875 or 5,948,648), peptidelinkers rich in proline, or peptide linkers having an inflexible helicalstructure. Rigid linkers are described in, for example, Chen, et al.(2013) Adv. Drug Deliv. Rev. 65(10):1357-1369; US 2010/0158823 and US2009/10221477.

In other embodiments, the linker is an in vivo cleavable linker. In vivocleavable linkers can include a cleavable disulfide bond formed betweentwo cysteine residues or linkers having a protease recognition sequence,e.g., recognized by matrix metalloproteases (MMPs).

Examples of suitable peptide linkers of use in the synthetic histatinare provided in Table 3.

TABLE 3 SEQ Type Sequence ID NO: Flexible (GGGGS)_(n)  3 FlexibleKESGSVSSEQLAQFRSLD  4 Flexible EGKSSGSGSESKST  5 Flexible GGGGGGGG  6Flexible GSAGSAAGSGEF  7 Flexible (GGSG)_(n)  8 Flexible (GS)_(n)  9Rigid (EAAAK)_(n) 10 Rigid A(EAAAK)_(n)A 11 Rigid PAPAP 12 Rigid(XP)_(n) 13 Cleavable VSQTSKLTRAETVFPDV 14 Cleavable PLGLWA 15 CleavableRVLAEA 16 Cleavable EDVVCCSMSY 17 Cleavable GGIEGRGS 18 CleavableTRHRQPRGWE 19 Cleavable AGNRVRRSVG 20 Cleavable RRRRRRRRR 21 CleavableGFLG 22 Cleavable CRRRRRREAEAC 23n is 1 to 5. X may be any amino acid residue, but is preferably Ala, Lysor Glu.

Each of the individual linkers, i.e., L₁ and L₂, of the synthetichistatin of this invention can be the same or different. In someembodiments, a synthetic histatin includes at least one flexible linker.In some embodiments, at least one flexible linker is a hydrocarbonlinker. In other embodiments, at least one flexible linker is a peptidelinker. In particular embodiments, each linker, i.e., L₁ and L₂, of thesynthetic histatin is a hydrocarbon linker. In certain embodiments, eachlinker, i.e., L₁ and L₂, of the synthetic histatin has the structure—(CH₂)₆—.

Examples of synthetic histatins containing combinations of metal bindingdomains and wound healing domains with flexible linkers are presented inTable 4.

TABLE 4 SEQ Synthetic Histatin ID NO: GYKRKFHEKHHSHR-(CH₂)₆-YGDYGSNYLYDN28 HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆- 29 YGDYGSNYLYDNHEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆- 30 YGDYGSNYLYDNHEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKHH-(CH₂)₆- 31 YGDYGSNYLYDNHEKRHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆- 32 YGDYGSNYLYDN

The synthetic histatins described herein are commonly referred to asfusion or chimeric polypeptides. Such molecules can be synthesized byroutine methods including recombinant protein expression, chemicalsynthesis, or a combination thereof. In some embodiments, the synthetichistatin of the invention is synthesized recombinantly using recombinantDNA techniques. Thus, the invention provides polynucleotides that encodethe synthetic histatin of the invention. In a related aspect, theinvention provides vectors, particularly expression vectors that harborthe polynucleotides encoding the synthetic histatin of the invention. Incertain embodiments, the vector provides replication, transcriptionand/or translation regulatory sequences that facilitate recombinantsynthesis of the desired synthetic histatin in a eukaryotic cell orprokaryotic cell. Accordingly, the invention also provides host cellsfor recombinant expression of the synthetic histatin and methods ofharvesting and purifying the synthetic histatin produced by the hostcells. Production and purification of recombinant peptides is a routinepractice to one of skilled in the art and any suitable methodology canbe used.

In another embodiment, the synthetic histatin is synthesized by any ofthe chemical synthesis techniques known in the art, particularlysolid-phase synthesis techniques, for example, usingcommercially-available automated peptide synthesizers. See, for example,Stewart & Young (1984) Solid Phase Peptide Synthesis, 2^(nd) ed., PierceChemical Co.; Tarn, et al. (1983) J. Am. Chem. Soc. 105:6442-55;Merrifield (1986) Science 232:341-347; and Barany et al. (1987) Int. J.Peptide Protein Res. 30:705-739.

The synthetic histatin can be isolated and/or purified by any suitablemethods known in the art including without limitation gel filtration andaffinity purification. In some embodiments, the synthetic histatin isproduced with a tag, e.g., an epitope tag, to facilitate isolation ofthe synthetic histatin. In one aspect, the synthetic histatin is atleast 1% pure, e.g., at least 5% pure, at least 10% pure, at least 20%pure, at least 40% pure, at least 60% pure, at least 80% pure, and atleast 90% pure, as determined by SDS-PAGE. Once isolated and/orpurified, the properties of the synthetic histatin can be readilyverified by techniques known to those skilled in the art.

While the HTN fragments disclosed herein are derived from humansequences, orthologs or allelic variants of the HTN fragments can alsobe used. The term “ortholog” refers to the same protein in anotherspecies (e.g., Macaca fascicularis, Trachypithecus cristatus,Chlorocebus aethiops, Nomascus leucogenys, or Gorilla gorilla), whichexhibits the same activity. By comparison, “allelic variant” refers thesame protein in the same species, which may have an altered amino acidsequence resulting from a polymorphism within the population. In certainembodiments, the HTN fragment ortholog or allelic variant has sequenceidentity of at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99%, but less than100%, to the HTN fragments disclosed herein.

Derivatives and analogs of the synthetic histatins described herein areall contemplated and can be made by altering their amino acid sequencesby substitutions, additions, and/or deletions/truncations or byintroducing chemical modifications that result in functionallyequivalent molecules. It will be understood by one of ordinary skill inthe art that certain amino acids in a sequence of any polypeptide may besubstituted for other amino acids without adversely affecting theactivity of the polypeptides.

In certain embodiments, the synthetic histatins of the invention includeone or more modifications including without limitation phosphorylation,glycosylation, hydroxylation, sulfonation, amidation, acetylation,carboxylation, palmitoylation, PEGylation, introduction ofnonhydrolyzable bonds, and disulfide formation. The modification mayimprove the stability and/or activity of the synthetic histatins.

For example, the C-terminal may be modified with amidation, addition ofpeptide alcohols and aldehydes, addition of esters, or addition ofp-nitroaniline and thioesters. The N-terminal and side chains may bemodified by PEGylation, acetylation, formylation, addition of a fattyacid, addition of benzoyl, addition of bromoacetyl, addition ofpyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl andaddition of 3-mercaptopropyl, acylations (e.g., lipopeptides),biotinylation, phosphorylation, sulfation, glycosylation, introductionof maleimido group, chelating moieties, chromophores or fluorophores.

In one embodiment, the synthetic histatin is conjugated to a fatty acid,e.g., the synthetic histatin is myristoylated. For example, a fatty acidmay be conjugated to the N-terminus of the synthetic histatin. Suchfatty acids include caprylic acid, capric acid, lauric acid, myristicacid, palmitic acid, stearic acid, etc. Furthermore, cysteines insynthetic histatin can be palmitoylated. In one embodiment, thesynthetic histatin is myristylated, stearylated or palmitoylated at theN terminal amino acid.

In addition, or as an alternative, to post-translational modifications,the synthetic histatin can be conjugated or linked to another peptide,such as a carrier peptide. The carrier peptide may facilitatecell-penetration and can include peptides such as antennapedia peptide,penetratin peptide, TAT, transportan or polyarginine. In an embodiment,the peptide is conjugated or linked to the antennapedia peptide,RQIKIWFQNRRMKWKK (SEQ ID NO:55).

A synthetic histatin of the invention may also be cyclized. As usedherein the term “cyclized” or “cyclic” denote an analog of a linearpeptide that incorporates at least one bridging group (e.g., an amide,thioether, thioester, disulfide, urea, carbamate, hydrocarbon orsulfonamide) between to amino acid residues to form a cyclic structure.The bridging group can present on the side chain of an amino acidresidue or a terminal amino acid residue thereby providing side chaincyclization (e.g., lactam bridge, thioester), head-to-tail cyclization,or hydrocarbon-stapled peptides.

In certain embodiments, the cyclic synthetic histatin has a disulfidebridge between two terminal cysteine residues. Representative amino acidsequences for preparing cyclized synthetic histatins are provided inTable 5.

TABLE 5 SEQ Cyclic Synthetic Histatin ID NO:CGYKRKFHEKHHSHR-(CH₂)₆-YGDYGSNYLYDNC 56CHEKHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆- 57 YGDYGSNYLYDNCCHEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆- 58 YGDYGSNYLYDNCCHEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKHH-(CH₂)₆- 59 YGDYGSNYLYDNCCHEKRHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆- 60 YGDYGSNYLYDNC

In other embodiments, the cyclic synthetic histatin is prepared from alinear peptide by cyclization with sortase. “Cyclization with sortase”or “cyclized with sortase” refers to a method of cyclizing a linearpeptide using the enzyme sortase. Sortase-based cyclization is known inthe art for manufacturing large cyclic peptides. See, Bolscher, et al.(2011) FASEB J. 25(8):2650-2658, and references cited therein.

Butelase cyclization has also been used to cyclize histatin. Addition ofthe tripeptide Asn-His-Val motif at the C-terminus provides a substratefor butelase to cyclize the synthetic histatin at a rate significantlyfaster than that of sortase A. See, Nguyen, et al. (2016) Nat. Protocols11:1977-88; Tam, et al. (June 2015) Peptides 2015: Proc. 24^(th) Am.Pept. Symp., Orlando, Fla., pg. 27.

One of skill in the art will recognize that the synthetic histatin ofthe invention will be beneficial for treating diseases. Accordingly, tofacilitate administration, this invention also provides a compositioncontaining one or more endogenous and/or synthetic histatins and apharmaceutically acceptable carrier or excipient. The pharmaceuticalcompositions provided herein can be formulated for oral, intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, intralesional or topical administration. Suitablepharmaceutical compositions can be determined by one skilled in the artdepending upon, for example, the intended route of administration,delivery format and desired dosage. See, for example, Remington'sPharmaceutical Sciences (19th edition, 1995).

The endogenous and/or synthetic histatin(s) can be incorporated in aconventional systemic dosage form, such as a tablet, capsule, softgelatin capsule, elixir or injectable formulation. The dosage forms mayalso include the necessary physiologically acceptable carrier material,excipient, lubricant, buffer, surfactant, antibacterial, bulking agent(such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) orthe like.

Acceptable formulation materials preferably are, nontoxic to recipientsat the dosages and concentrations employed. The pharmaceuticalcomposition may contain formulation materials for modifying, maintainingor preserving, for example, the pH, osmolarity, viscosity, clarity,color, isotonicity, odor, sterility, stability, rate of dissolution orrelease, adsorption or penetration of the composition. Suitableformulation materials include, but are not limited to, amino acids (suchas glycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl,citrates, phosphates or other organic acids); bulking agents (such asmannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides,disaccharides, and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid, phenethylalcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid orhydrogen peroxide); solvents (such as glycerin, propylene glycol orpolyethylene glycol); sugar alcohols (such as mannitol or sorbitol);suspending agents; surfactants or wetting agents (such as PLURONICS,PEG, sorbitan esters, polysorbates such as polysorbate 20 andpolysorbate 80, TRITON, trimethamine, lecithin, cholesterol, ortyloxapal); stability enhancing agents (such as sucrose or sorbitol);tonicity enhancing agents (such as alkali metal halides, preferablysodium or potassium chloride, mannitol, or sorbitol); delivery vehicles;diluents; excipients and/or pharmaceutical adjuvants. See, for example,Remington's Pharmaceutical Sciences, Id.

The primary carrier or excipient in a pharmaceutical composition may beeither aqueous or nonaqueous in nature. For example, a suitable carrieror excipient may be water for injection, physiological saline solutionor artificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. Neutralbuffered saline or saline mixed with serum albumin are further exemplaryexcipients. Pharmaceutical compositions can include Tris buffer of aboutpH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may furtherinclude sorbitol or a suitable substitute. Pharmaceutical compositionsof the invention may be prepared for storage by mixing the selectedcomposition having the desired degree of purity with optionalformulation agents (Remington's Pharmaceutical Sciences, Id.) in theform of a lyophilized cake or an aqueous solution. Further, theendogenous or synthetic histatins of the invention may be formulated asa lyophilizate using appropriate excipients such as sucrose.

Administration routes for the pharmaceutical compositions of theinvention include the oral route; injection by intravenous,intraperitoneal, intracerebral (intra-parenchymal),intracerebroventricular, intramuscular, intra-ocular, intraarterial,intraportal, or intralesional routes; or via sustained release systemsor by implantation devices. The pharmaceutical compositions may beadministered by bolus injection or continuously by infusion, or byimplantation device. The pharmaceutical composition also can beadministered locally via implantation of a membrane, sponge or anotherappropriate material onto which the synthetic histatin(s) has beenabsorbed or encapsulated. Where an implantation device is used, thedevice may be implanted into any suitable tissue or organ, and deliveryof the endogenous or synthetic histatin(s) may be via diffusion,timed-release bolus, or continuous administration.

When parenteral administration is contemplated, the compositions for usein this invention may be in the form of a pyrogen-free, parenterallyacceptable aqueous solution containing the endogenous or synthetichistatin(s) of the invention in a pharmaceutically acceptable vehicle. Aparticularly suitable vehicle for parenteral injection is steriledistilled water in which the endogenous or synthetic histatin(s) isformulated as a sterile, isotonic solution, appropriately preserved.Preparation can involve the formulation of the synthetic histatin(s)with an agent, such as injectable microspheres, bio-erodible particles,polymeric compounds (such as polylactic acid or polyglycolic acid),beads or liposomes, that may provide controlled or sustained release ofthe synthetic histatin(s), which may then be delivered via a depotinjection. In particular, formulation with hyaluronic acid has theeffect of promoting sustained duration in the circulation.

The compositions may also be formulated for inhalation. In theseembodiments, the endogenous or synthetic histatin(s) of the invention isformulated as a dry powder for inhalation, or inhalation solutions mayalso be formulated with a propellant for aerosol delivery, such as bynebulization. Pulmonary administration is further described in, e.g., WO1994/020069.

The pharmaceutical compositions of the invention can be deliveredthrough the digestive tract, such as orally. The preparation of suchpharmaceutically acceptable compositions is within the skill of the art.The endogenous or synthetic histatin(s) of the invention that isadministered in this fashion may be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. A capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe endogenous or synthetic histatin(s). Diluents, flavorings, lowmelting point waxes, vegetable oils, lubricants, suspending agents,tablet disintegrating agents, and binders may also be used.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms can be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid and the like. It may also be desirableto include isotonic agents such as sugars, sodium chloride and the like.Prolonged absorption of an injectable pharmaceutical form can be broughtabout by the inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

In certain embodiments, the endogenous or synthetic histatin(s) isformulated for treating ocular diseases or conditions, both on thesurface and inside the eye. In particular embodiments, the endogenous orsynthetic histatin(s) of the invention may be formulated andadministered to the eye in drop form; topical gel form; as a solidformulation (e.g., similar to LACRISERT, hydroxypropyl celluloseophthalmic insert); by injection into the anterior chamber of the eye;by injection into posterior chamber of the eye for inhibition ofangiogenesis, inhibition of destructive MMP activity or to enhanceepithelial wound healing; by coating of surgical devices (intraocularlens, glaucoma device, keratoprosthetic, lacrimal intubation tubes,lacrimal bypass tubes); by coating of contact lenses; or by coating ofmicrobeads, nanobeads or other similar constructs.

As one skilled in the art will also appreciate, the compositiondescribed herein can be formulated so as to carry a minimum of adverseside effects. The compositions described herein can be suitable for longterm use alone; useful as an adjunct therapy along with NSAIDS,glucocorticoids, immunosuppressive agents or anti-angiogenic agents;and/or useful in a program involving rotation between any or all ofthese agents, thereby decreasing long term exposure to (and, therefore,side effects resulting from) any one agent.

This invention also provides kits containing one or more of theendogenous or synthetic histatins, or a pharmaceutical compositioncontaining the same, and optionally one or more NSAIDS, glucocorticoids,immunosuppressive agents or anti-angiogenic agents. Kits are typicallyprovided in a suitable container (e.g., for example, a foil, plastic, orcardboard package). In certain embodiments, a kit may include one ormore pharmaceutical excipients or carriers, pharmaceutical additives,and the like, as is described herein. In other embodiments, a kit mayinclude a means for proper administration, such as, for example,graduated cups, syringes, needles, cleaning aids, an intraocular lens, aglaucoma device, an orbital implant, keratoprosthetic, lacrimalintubation tubes, lacrimal bypass tube, a contact lens and the like. Incertain embodiments, a kit may include instructions for properadministration and/or preparation for proper administration.

Given the antimicrobial, wound healing, metal ion chelating,anti-inflammatory, anti-cancer, anti-angiogenic, and matrixmetalloproteinase inhibitory applications of histatins, this inventionalso provides methods of treating a subject having a disease orcondition for which histatins would provide a benefit. In accordancewith such methods, a subject is administered one or more the synthetichistatins and/or endogenous histatins of this invention in an amounteffective to treat the disease or condition. “Subject,” as used herein,is meant to include humans, as well as non-human animals, particularlythose who suffer from or who are susceptible to developing one or moreof the disease or conditions described herein.

As used herein, the term “effective amount” or “therapeuticallyeffective amount” refers to an amount of an endogenous or synthetichistatin of the invention or a pharmaceutical composition containing theendogenous or synthetic histatin sufficient to achieve the stateddesired result. The amount of the peptide which constitutes an“effective amount” or “therapeutically effective amount” may varydepending on the severity of the disease, the condition, weight, or ageof the patient to be treated, the frequency of dosing, or the route ofadministration, but can be determined routinely by one of ordinary skillin the art. A clinician may titer the dosage or route of administrationto obtain the optimal therapeutic effect. Typical dosages range fromabout 0.1 μg/kg to up to about 100 mg/kg or more, depending on thefactors mentioned above. In certain embodiments, the dosage may rangefrom 0.1 μg/kg up to about 100 mg/kg, or 1 μg/kg up to about 100 mg/kg,or 5 μg/kg up to about 100 mg/kg.

“Treating” a subject having a disease or condition means accomplishingone or more of the following: (a) reducing the severity of the diseaseor condition; (b) arresting the development of the disease or condition;(c) inhibiting worsening of the disease or condition; (d) limiting orpreventing recurrence of the d disease or condition in patients thathave previously had the disease or condition; (e) causing regression ofthe disease or condition; (f) improving or eliminating the symptoms ofthe disease or condition; and/or (g) improving survival. As such, thetreatment methods of this invention encompass both therapeutic andprophylactic administration.

In accordance with this invention, endogenous or synthetic histatins areof particular use in the treatment of ocular diseases or conditionsincluding, but not limited to, ocular surface inflammatory disorderssuch as lupus, rheumatoid arthritis, corneal inflammation (e.g.,Mooren's or inflammatory and infectious ulcerations), necrotizingscleritis, sarcoidosis or Wegener's disease; age-related maculardegeneration (wet or dry); diabetic retinopathy; chronic or acute severeuveitis; retinal pigment epithelial disorders, e.g., hereditarydisorders such as retinitis pigmentosa as well as non-hereditary retinaldegenerations; ocular surface diseases mediated by inflammation, e.g.,dry eye disease, graft versus host disease, Stevens-Johnson syndrome,alkali burns, and chronic atopic diseases such as atopic or allergicconjunctivitis or eczematous diseases; fungal and bacterial infection;corneal and conjunctival wounds, in particular wounds associated withneurotrophic/diabetic neuropathy. As a result of endogenous or synthetichistatin administration, corneal and scleral damage and melting aredecreased and the activity of endogenous or environmental inflammatoryagents, e.g., LPS (bound by histatin) are reduced.

In addition to the treatment of ocular diseases or conditions, theendogenous or synthetic histatins can be tailored to promote woundhealing, and/or provide antimicrobial, metal ion chelating,anti-inflammatory, anti-angiogenic, and/or matrix metalloproteinaseinhibitory activity in other tissues or organs of interest. In oneembodiment, lamellar tissues, nerve tissues, connective tissues,vascular tissues, muscle tissues, skeletal tissues, or blood componentsare treated. In another embodiment, organs such as skin, liver, lung,kidney, heart, or bowel are treated. Treatment of such tissues andorgans with an endogenous or synthetic histatin may provide benefit forsubjects with wounds, infection, inflammatory and/or degenerativeconditions such as atherosclerosis and arteriosclerosis, osteoarthritisand other degenerative joint diseases, optic atrophy, musculardystrophy, degenerative processes associated with aging, asthma,dermatitis, laminitis, reactive airway disease, inflammatory boweldisease, multiple sclerosis, periodontal disease, psoriasis, type Idiabetes, and ischemia-reperfusion injury.

The following non-limiting examples are provided to further illustratethe present invention.

Example 1: Histatin Synthesis and Purification

Linear peptide was synthesized using the stepwise solid-phase method bythe 9-fluorenylmethoxycarbonyl (Fmoc) chemistry on the Wang resin(AnaSpec, Fremont, Calif.) with a 12 channel multiplex peptidesynthesizer (Protein Technologies, Tucson, Ariz.) according to themanufacturer's procedures. Peptide synthesis started from the C-terminusof the peptide. The Fmoc group of the resin was removed with 20%piperidine in N,N-dimethylformamide (DMF) (5 min, ×2) followed bywashing the resin with DMF (30 sec, 6×) before the amino acid (Fmocprotected, 2 equiv) was added in the presence of 0.2 M2-(1H-Benzotriazole-l-yl)-1,1,3,3,-tetramethyluroniumhexafluorophosphate (HBTU, 1.9 equiv) and 0.4 M 4-methylmorpholine (NMM,4 equiv) in DMF (30 min, ×3). Excess reagents were washed away (30 sec,6×) with DMF. The process was repeated until the last amino acid wasadded. After completion, the N-terminal Fmoc was removed with 20%piperidine in DMF (5 min, ×2) followed by washing the resin with DMF (30sec, 6×). Detachment of peptide from the resin and removal of the sidechain protection groups were done by incubating the resin withtrifluoroacetic acid (TFA):Thioanisole:Water:Phenol:1,2-ethanedithio(82.5:5:5:5:2.5 v/v) cocktail for 2 hours. The reaction mixture wasfiltered followed by washing the resin with TFA (2×). Ice-cold ethylether was added to precipitate the peptide and the pellet was washed 2times with ice-cold ethyl ether. The crude peptide was then dissolved in50% acetonitrile in water and lyophilized.

The crude peptide was purified on a preparative KINETEX reversed-phaseC18 column, 150×21.1 mm (Phenomenex, Torrance, Calif.) using a BIOCADSPRINT HPLC system (Applied Biosystems, Foster City, Calif.). A flowrate of 30 mL/min with solvent A (0.1% TFA in deionized water) andsolvent B (0.1% TFA in acetonitrile) was used. The column wasequilibrated with 5% solvent B before sample injection. Elution wasperformed with a linear gradient from 5% solvent B to 100% solvent B in60 min. The absorbance of the column effluent was monitored at 214 nm,and peak fractions were pooled and lyophilized. The pure peptidefraction was identified by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) or electrosprayionization mass spectrometry (ESI MS) and lyophilized.

Example 2: Matrix Metalloproteinase Inhibition

Zymography analysis of a synthetic histatin containing three metalbinding domains from HTN5 and one wound healing domain (i.e., SEQ IDNO:29) was carried out using conventional methods. See, e.g., Thomadaki,et al. (2013) Oral Dis. 19(8):781-8. This analysis demonstrated that thesynthetic histatin inhibited MMP2 activity in a dose-dependent manner.See FIG. 1.

Example 3: Wound Healing Activity

A conventional in vitro scratch assay was used to measure cell migrationin the presence of a synthetic histatin containing three metal bindingdomains from HTN5 and one wound healing domain (i.e., SEQ ID NO:29). A“scratch” was made in a monolayer of human corneal epithelium cells andtime lapsed microscopy was used to capture images at the beginning andat regular intervals during cell migration to close the scratch. Theresults of this analysis indicated a notable enhancement of woundhealing as evidenced by a reduction in time to wound closure (Table 6).

TABLE 6 Amount of Synthetic Histatin Time to Closure Control (0 μM) Noclosure in 48 hours  1 μM 48 hours 10 μM 22 hours

Example 4: Anti-Angiogenesis Activity

Tube formation assays were used to assess the anti-angiogenic propertyof synthetic histatin compared to native HTN1 and HTN2. Human umbilicalvein endothelial cells (HUVECs) were suspended in diluted MATRIGEL foran overnight incubation and then subjected to a media change containingVEGF (10 ng/mL) (Goodwin (2007) Microvasc. Res. 74:172-83) alone or VEGF(10 ng/mL) in combination with HTN1, HTN2 or synthetic histatin (50 μM).While capillary-like structures were apparent when cells were treatedwith VEGF only, cells treated with synthetic histatin did not form suchstructures (FIG. 2).

Example 5: Changes in Gene Expression

Pathophysiological analyses of dry eye disease indicate that thecritical factors involved in the development of dry eye disease involveinflammatory pathways including Th17/IL-17, I-CAM, IL-8,TNFα/IL-1/IL-6/NFκB, IL-10, MMP9 (and other matrix metalloproteinases)and TLR4/HSP (Stevenson, et al. (2012) Arch. Ophthalmol. 130(1):90-100).In addition, it has been reported that Ephrin A is involved in thedevelopment of epitheliopathy, poor wound healing and corneal diseaserelated to elevated sugar levels/diabetic and neuropathic ulcers of theocular surface. Accordingly, it was determined what effect synthetichistatin application would have on the gene expression in human cornealepithelium. With regard to inflammatory pathways, this analysisindicated that 7 of the 44 genes in the Th17/IL-17 pathway exhibitedsignificant changes in gene expression in response to synthetic histatintreatment. Th17/IL-17 pathway is a central and important pathway in dryeye disease (Stevenson, et al. (2012) Arch Ophthalmol. 130(1):90-100).

Further, a 333% reduction in I-CAM gene expression and 400% reduction inthe levels of IL-8 were observed. Moreover, 6 of the 62 genes in theIL-10 pathway were directed affected and 5 of 54 genes in the TLR4/HSPpathway were significantly affected. In addition, expression of severalmembers of the TNFα/IL-1/IL-6/NFKB pathway were significant reduced andexpression of genes related to MMP activity were reduced. These geneexpression changes demonstrate the reduction in expression ofinflammatory genes strongly associated with dry eye disease and ocularinflammatory diseases. Consistent with an improvement in cornealepithelial migration, path finding, cell spreading and wound closure, a260% reduction in levels of Ephrin A was observed in response tosynthetic histatin treatment.

Example 6: Treatment of Dry Eye Disease

Histatins are a histidine rich family of proteins which have primarilybeen found in saliva. These endogenous proteins (see Table 1), inaddition to the synthetic histatins described herein, may have a numberof therapeutic applications through a number of different means ofdelivery (e.g., topical, systemic, injected, carried on deliverymaterials). Such applications may include enhancement of ocular surfacewound healing, prevention of ocular surface or intraocular inflammationand angiogenesis and prevention or treatment of dry eye diseases(inflammatory or otherwise).

Dry eye is a multifactorial disorder of the tears and ocular surfacethat results in symptoms of discomfort, visual disturbance, and tearfilm instability, with potential damage to the ocular surface. It isaccompanied by increased osmolarity of the tear film and inflammation ofthe ocular surface (Lemp (1995) CLAO J. 21:221-2). For a more detaileddefinition, see “The definition and classification of dry eye disease:report of the Definition and Classification Subcommittee of theInternational Dry Eye WorkShop” (2007) Ocul. Surf. 5(2):75-9.

One particularly useful model of dry eye disease and inflammatory oculardisease is the use of benzalkonium chloride (BAK) to induce cell death,inflammation and injury to ocular surfaces and corneal cells (Tressler,et al. (2011) Ocul. Surf. 9:140-58; Paimela, et al. (2012) Mol. Vis.18:1189-1196). Briefly, human corneal epithelial cells were culturedunder standard conditions. Cells were treated with native HTN5 (50 μM)or synthetic histatin (10 μM) in the presence or absence of 0.002% BAK.The permeability of cellular membranes following the exposures wasdetermined by measuring the amount of released lactate dehydrogenase(LDH) enzyme from the cells. Maximum LDH release of cells was determinedby lysing cells, and subsequently measuring the LDH from the culturemedium. As shown in FIG. 3, topical HTN5 and synthetic histatin provideda statistically significant reduction in cell death. Accordingly,endogenous and synthetic histatins could be used to protect cells frominjury, inflammation and cell death and are therefore of use in thetreatment of dry eye disease.

What is claimed is:
 1. A method of treating an ocular disease orcondition comprising administering to a subject in need of treatment aneffective amount of a synthetic histatin having the general structure:[HTNF₁-L₁-HTNF₂-(L₂)_(y)]_(x)  (Formula I) wherein i) HTNF₁ is a firsthistatin fragment ranging in length from 5 to 20 amino acids; ii) HTNF₂is a second histatin fragment ranging in length from 5 to 20 aminoacids; iii) L₁ is a first linker; iv) L₂ is a second linker; v) x=1 to3; and vi) y=0 to 2; wherein HTNF₁ and HTNF₂ are each independently thesame or different and at least one of L₁ or L₂ is a hydrocarbon linker,thereby treating the subject's ocular disease or condition.
 2. Themethod of claim 1, wherein HTNF₁ or HTNF₂ has an amino acid sequence ofSNYLYDN (SEQ ID NO:1) or HEXXH (SEQ ID NO:2), wherein each X isindependently a basic amino acid residue.
 3. The method of claim 1,wherein the other of L₁ or L₂ is a flexible linker, rigid linker, invivo cleavable linker, or a combination thereof.
 4. The method of claim3, wherein the flexible linker is a hydrocarbon linker.
 5. The method ofclaim 4, wherein the hydrocarbon linker has the structure —(CH₂)₆—. 6.The method of claim 3, wherein the flexible linker is a peptide linkerhaving an amino acid sequence of (GGGGS)_(n) (SEQ ID NO:3),KESGSVSSEQLAQFRSLD (SEQ ID NO:4), or EGKSSGSGSESKST (SEQ ID NO:5),GGGGGGGG (SEQ ID NO:6), GSAGSAAGSGEF (SEQ ID NO:7), (GGSG)_(n) (SEQ IDNO:8), or (GS)_(n) (SEQ ID NO:9), wherein n is 1, 2, 3, 4 or
 5. 7. Themethod of claim 3, wherein the rigid linker is a peptide linker havingan amino acid sequence of (EAAAK)_(n) (SEQ ID NO:10), A(EAAAK)_(n)A (SEQID NO:11), PAPAP (SEQ ID NO:12), or (XP)_(n) (SEQ ID NO:13), wherein nis 1, 2, 3, 4 or
 5. 8. The method of claim 3, wherein the in vivocleavable linker is a peptide linker having an amino acid sequence ofVSQTSKLTRAETVFPDV (SEQ ID NO:14), PLGLWA (SEQ ID NO:15), RVLAEA (SEQ IDNO:16), EDVVCCSMSY (SEQ ID NO:17), GGIEGRGS (SEQ ID NO:18), TRHRQPRGWE(SEQ ID NO:19), AGNRVRRSVG (SEQ ID NO:20), RRRRRRRRR (SEQ ID NO:21),GFLG (SEQ ID NO:22), or CRRRRRREAEAC (SEQ ID NO:23).
 9. The method ofclaim 1, wherein said synthetic histatin is linear or cyclized.
 10. Themethod of claim 9, wherein the synthetic histatin is cyclized via adisulfide bridge between terminal cysteine residues.
 11. The method ofclaim 9, wherein the synthetic histatin is cyclized with a sortase orbutelase.
 12. The method of claim 1, wherein said synthetic histatin hasthe structure: (a) GYKRKFHEKHHSHR(SEQ ID NO: 24)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25); (b) HEKHH(SEQ ID NO: 26)-L₁-HEKHH(SEQ ID NO: 26)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25);(c) HEKRHH(SEQ ID NO: 27)-L₁-HEKRHH(SEQ ID NO:27)-L₂-HEKRHH(SEQ ID NO: 27)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25);(d) HEKRHH(SEQ ID NO: 27)-L₁-HEKRHH(SEQ ID NO: 27)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25); or(e) HEKRHH(SEQ ID NO: 27)-L₁-HEKHH(SEQ ID NO: 26)-L₂-HEKHH(SEQ ID NO: 26)-L₁-YGDYGSNYLYDN(SEQ ID NO: 25),

wherein at least one of L₁ or L₂ is a hydrocarbon linker and the otherof L₁ or L₂ is a flexible linker, rigid linker, or in vivo cleavablelinker.
 13. The method of claim 1, wherein said synthetic histatin hasthe structure: (a) (SEQ ID NO: 28) GYKRKFHEKHHSHR-(CH₂)₆-YGDYGSNYLYDN;(b) (SEQ ID NO: 29) HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-YGDYGSNYLYDN; (c) (SEQ ID NO: 30)HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆- YGDYGSNYLYDN; (d) (SEQ ID NO: 31) HEKRHH-(CH₂)₆-HEKRHH-(CH₂)₆-HEKHH-(CH₂)₆- YGDYGSNYLYDN;or (e) (SEQ ID NO: 32) HEKRHH-(CH₂)₆-HEKHH-(CH₂)₆-HEKHH-(CH₂)₆-YGDYGSNYLYDN


14. The method of claim 1, wherein the ocular disease or condition is anocular surface inflammatory disorder, dry eye disease, ocular tissueneovascularization, age-related macular degeneration, diabeticretinopathy, chronic or acute severe uveitis, a retinal pigmentepithelial disorder, an ocular surface disease, an infection, or cornealor conjunctival wound.
 15. The method of claim 1, wherein the synthetichistatin provides wound healing, antimicrobial, metal ion chelating,anti-inflammatory, anti-angiogenic, or matrix metalloproteinaseinhibitory activity.
 16. A method of treating an ocular disease orcondition comprising administering to a subject in need of treatment aneffective amount of a synthetic histatin having the general structure:[HTNF₁-L₁-HTNF₂-(L₂)_(y)]_(x)  (Formula I) wherein i) HTNF₁ is a firsthistatin fragment ranging in length from 5 to 20 amino acids; ii) HTNF₂is a second histatin fragment ranging in length from 5 to 20 aminoacids; iii) L₁ is a first linker; iv) L₂ is a second linker; v) x=1 to3; and vi) y=0 to 2; wherein HTNF₁ and HTNF₂ are each independently thesame or different and L₁ and L₂ are each independently the same ordifferent, and wherein at least one of HTNF₁ or HTNF₂ is HEXXH (SEQ IDNO:2) or HEKRHH (SEQ ID NO:27), wherein each X is independently a basicamino acid residue, and wherein when each of HTNF₁ and HTNF₂ is HEHKH(SEQ ID NO:50), x is 1 and y is 0, thereby treating the subject's oculardisease or condition.
 17. The method of claim 16, wherein HTNF₁ or HTNF₂has an amino acid sequence of SNYLYDN (SEQ ID NO:1).
 18. The method ofclaim 16, wherein L₁ or L₂ is a flexible linker, rigid linker, in vivocleavable linker, or a combination thereof.
 19. The method of claim 18,wherein the flexible linker is a hydrocarbon linker comprising 3 to 25methylene groups.
 20. The method of claim 19, wherein the hydrocarbonlinker has the structure —(CH₂)₆—.
 21. The method of claim 18, whereinthe flexible linker is a peptide linker having an amino acid sequence of(GGGGS)_(n) (SEQ ID NO:3), KESGSVSSEQLAQFRSLD (SEQ ID NO:4), orEGKSSGSGSESKST (SEQ ID NO:5), GGGGGGGG (SEQ ID NO:6), GSAGSAAGSGEF (SEQID NO:7), (GGSG)_(n) (SEQ ID NO:8), or (GS)_(n) (SEQ ID NO:9), wherein nis 1, 2, 3, 4 or
 5. 22. The method of claim 18, wherein the rigid linkeris a peptide linker having an amino acid sequence of (EAAAK)_(n) (SEQ IDNO:10), A(EAAAK)_(n)A (SEQ ID NO:11), PAPAP (SEQ ID NO:12), or (XP)_(n)(SEQ ID NO:13), wherein n is 1, 2, 3, 4 or
 5. 23. The method of claim18, wherein the in vivo cleavable linker is a peptide linker having anamino acid sequence of VSQTSKLTRAETVFPDV (SEQ ID NO:14), PLGLWA (SEQ IDNO:15), RVLAEA (SEQ ID NO:16), EDVVCCSMSY (SEQ ID NO:17), GGIEGRGS (SEQID NO:18), TRHRQPRGWE (SEQ ID NO:19), AGNRVRRSVG (SEQ ID NO:20),RRRRRRRRR (SEQ ID NO:21), GFLG (SEQ ID NO:22), or CRRRRRREAEAC (SEQ IDNO:23).
 24. The method of claim 16, wherein said synthetic histatin islinear or cyclized.
 25. The method of claim 24, wherein the synthetichistatin is cyclized via a disulfide bridge between terminal cysteineresidues.
 26. The method of claim 24, wherein the synthetic histatin iscyclized with a sortase or butelase.
 27. The method of claim 16, whereinthe ocular disease or condition is an ocular surface inflammatorydisorder, dry eye disease, ocular tissue neovascularization, age-relatedmacular degeneration, diabetic retinopathy, chronic or acute severeuveitis, a retinal pigment epithelial disorder, an ocular surfacedisease, an infection, or corneal or conjunctival wound.
 28. The methodof claim 16, wherein the synthetic histatin provides wound healing,antimicrobial, metal ion chelating, anti-inflammatory, anti-angiogenic,or matrix metalloproteinase inhibitory activity.